U.S. patent number 6,673,569 [Application Number 09/393,171] was granted by the patent office on 2004-01-06 for dsba/dsbb/dsbc/dsbd expression plasmid.
This patent grant is currently assigned to HSP Research Institute, Inc.. Invention is credited to Yoichi Kurokawa, Hideki Yanagi, Takashi Yura.
United States Patent |
6,673,569 |
Kurokawa , et al. |
January 6, 2004 |
DsbA/DsbB/DsbC/DsbD expression plasmid
Abstract
An artificial operon comprising polynucleotides encoding each of
DsbA, DsbB, DsbC and DsbD; an expression plasmid carrying the above
artificial operon, usable for expression of DsbA, DsbB, DsbC and
DsbD; a cotransformant harboring the above expression plasmid and
an expression vector for a foreign protein; and a method for
producing a foreign protein comprising culturing the
cotransformant.
Inventors: |
Kurokawa; Yoichi (Kyoto,
JP), Yanagi; Hideki (Takarazuka, JP), Yura;
Takashi (Kyoto, JP) |
Assignee: |
HSP Research Institute, Inc.
(Osaka, JP)
|
Family
ID: |
17282453 |
Appl.
No.: |
09/393,171 |
Filed: |
September 9, 1999 |
Foreign Application Priority Data
|
|
|
|
|
Sep 9, 1998 [JP] |
|
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10-255702 |
|
Current U.S.
Class: |
435/69.1;
435/69.7; 536/23.5; 536/23.6; 536/23.7 |
Current CPC
Class: |
C07K
1/1133 (20130101); C07K 14/245 (20130101); C12N
9/0051 (20130101) |
Current International
Class: |
C07K
14/245 (20060101); C07K 1/00 (20060101); C07K
1/113 (20060101); C07K 14/195 (20060101); C12N
9/02 (20060101); C12P 021/06 () |
Field of
Search: |
;435/69.1,69.4,69.5,69.51,69.52,69.6,70.1,71.1,71.2,320.1,455,465
;536/23.7,23.5,23.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
A Rietsch et al., Proc.Natl.Acad.Sci USA, "An in vivo pathway for
disulfide bond isomerization in Escherichia coli," Nov. 1996, vol.
93, pp. 13048-13053.* .
R Metheringham et al., Mol.Gen Genet, "Effects of mutations in
genes for proteins involved in disulphide bond formation in the
periplasm on the activities of anaerobically induced electron
transfer chains in Escherichia coli K12," 1996, 253:95-102.* .
D Missiakas et al., "Identification and characterization of a new
disulfide isomerase-like protein (DsbD) in Escherichia coli," pp.
3415-3424.* .
Dominique Missiakas et al., EMBO Journal, vol. 14, No. 14, pp.
3415-3424 (1995). .
Satoshi Kishigami et al., Genes to Cells, vol. 1, pp. 201-208
(1996). .
R. Metheringham et al., Molecular and General Genetics, vol. 253,
No. 1-2, pp. 95-102 (1996). .
J.C. Bardwell, Mol. Microbiol. 14(2),pp. 199-205 (1994). .
M. Sone et al., J. Biol. Chem., 272(16),pp. 10349-10352 (1997).
.
A. Rietsch et al., Proc. Natl. Acad. Sci. USA, 93, pp. 13048-13053
(1996). .
A. Knappik et al., Bio/Technol., 11, 77-83 (1993). .
M. Wunderlich et al., J. Biol. Chem., 268(33),pp. 24547-24550
(1993). .
C. Wulfing et al., J. Mol. Biol., 242, pp. 655-669 (1994). .
J. C. Joly et al., Proc. Natl. Acad. Sci. USA, 95, pp. 2773-2777
(1998). .
S. Kamitani et al., EMBO J., 11(1), pp. 57-62 (1992). .
S. Kishigami et al., Genes to Cells, 1, pp. 201-208 (1996). .
Y. Kohara et al., Cell, 50, pp. 495-508 (1987). .
J. Perez et al., Gene, 158, pp. 141-142 (1995). .
D. Koshland et al., Cell, 20, pp. 749-760 (1980)..
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Primary Examiner: Crouch; Deborah
Assistant Examiner: Woitach; Joseph
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Claims
What is claimed is:
1. An artificial operon comprising: (a) a polynucleotide comprising
a nucleic acid sequence which encodes a polypeptide (DsbA)
comprising an amino acid sequence set forth in SEQ ID NO:1, or (b)
a polynucleotide comprising a nucleic acid sequence encoding DsbA
that hybridizes under stringent conditions to the complement of SEQ
ID NO:1, wherein the polypeptide encoded by the polynucleotide
forms disulfide bonds in a nascent polypeptide chain which has been
transferred into the periplasm; (c) a polynucleotide comprising a
nucleic acid sequence which encodes a polypeptide (DsbB) comprising
an amino acid sequence set forth in SEQ ID NO:3, or (d) a
polynucleotide comprising a nucleic acid sequence encoding DsbB
that hybridizes under stringent conditions to the complement of SEQ
ID NO:3, wherein the polypeptide encoded by the polynucleotide
reoxidizes DsbA; (e) a polynucleotide comprising a nucleic acid
sequence which encodes a polypeptide (DsbC) comprising an amino
acid sequence set forth in SEQ ID NO:5, or (f) a polynucleotide
comprising a nucleic acid sequence encoding DsbC that hybridizes
under stringent conditions to the complement of SEQ ID NO:5,
wherein the polypeptide encoded by the polynucleotide corrects the
disulfide by means of cleavage of the disulfide bonds followed by
re-crosslinking; and (g) a polynucleotide comprising a nucleic acid
sequence which encodes a polypeptide (DsbD) comprising an amino
acid sequence set forth in SEQ ID NO:7, or (h) a polynucleotide
comprising a nucleic acid sequence encoding DsbD that hybridizes
under stringent conditions to the complement of SEQ ID NO:7,
wherein the polypeptide encoded by the polynucleotide reduces
DsbC.
2. The artificial operon according to claim 1, further comprising
an inducible promoter, said promoter capable of expressing each of
DsbA, DsbB, DsbC, and DsbD.
3. The artificial operon according to claim 2, wherein said
inducible promoter is selected from the group consisting of lac,
tac, trc, trp, ara, Pzt-1 and T7.
4. An artificial operon comprising a polynucleotide encoding DsbC
and a polynucleotide encoding DsbD, wherein the polynucleotide
encoding DsbC is selected from the group consisting of (e) a
polynucleotide comprising a nucleic acid sequence which encodes a
polypeptide comprising an amino acid sequence of SEQ ID NO: 5
(DsbC), or (f) a polynucleotide comprising a nucleic acid sequence
that specifically hybridizes under stringent conditions to the
complement of (e), wherein the polypeptide encoded by the
polynucleotide corrects the disulfide by means of cleavage of the
disulfide bonds followed by re-crosslinking; and the polynucleotide
encoding DsbD is selected from the group consisting of (g) a
polynucleotide comprising a nucleotide sequence which encodes a
polypeptide comprising an amino acid sequence of SEQ ID NO:7
(DsbD), or (h) a polynucleotide comprising a nucleic acid sequence
that specifically hybridizes to the complement of (g), wherein the
polypeptide encoded by the polynucleotide re-reduces DsbC.
5. An expression plasmid comprising the artificial operon according
to any one of claims 1, 2, 4.
6. A costransformant obtainable by introducing both the expression
plasmid according to claim 5 and an expression vector comprising a
polynucleotide encoding a foreign protein into a host cell.
7. The cotransformant according to claim 6, wherein the host cell
is an E. coli host cell.
8. The cotransformant according to claim 7, wherein the E. coli
host cell is an E. coli protease mutant.
9. The cotransformant according to claim 7, wherein said foreign
protein is selected from the group consisting of interferons,
interleukins, interleukin receptors, interleukin receptor
antagonists, granulocyte colony-stimulating factors, granulocyte
macrophage colony-stimulating factors, macrophage
colony-stimulating factors, erythropoietin, thrombopoietin,
leukemia inhibitors, stem cell growth factors, tumor necrosis
factors, growth hormones, proinsulin, insulin-like growth factors,
fibroblast growth factors, platelet-derived growth factors,
transforming growth factors, hepatocyte growth factors, bone
morphogenetic factors, nerve growth factors, ciliary neurotropic
factors, brain-derived neurotrophic factors, glia cell line-derived
neurotrophic factors, neurotrophin, angiogenesis inhibitors,
prourokinase, tissue plasminogen activators, blood coagulation
factors, protein C, glucocerebrosidase, superoxide dismutase,
renin, lysozyme, P450, prochymosin, trypsin inhibitors, elastase
inhibitors, lipocortin, leptin, immunoglobulins, single-chain
antibodies, complement components, serum albumin, cedar pollen
allergens, hypoxia-induced stress proteins, protein kinases,
proto-oncogene products, transcription factors and
virus-constitutive proteins.
10. The cotransformant according to claim 6, wherein said foreign
protein is selected from the group consisting of interferons,
interleukins, interleukin receptors, interleukin receptor
antagonists, granulocyte colony-stimulating factors, granulocyte
macrophage colony-stimulating factors, macrophage
colony-stimulating factors, erythropoietin, thrombopoietin,
leukemia inhibitors, stem cell growth factors, tumor necrosis
factors, growth hormones, proinsulin, insulin-like growth factors,
fibroblast growth factors, platelet-derived growth factors,
transforming growth factors, hepatocyte growth factors, bone
morphogenetic factors, nerve growth factors, ciliary neurotropic
factors, brain-derived neurotrophic factors, glia cell line-derived
neurotrophic factors, neurotrophin, angiogenesis inhibitors,
prourokinase, tissue plasminogen activators, blood coagulation
factors, protein C, glucocerebrosidase, superoxide dismutase,
renin, lysozyme, P450, prochymosin, trypsin inhibitors, elastase
inhibitors, lipocortin, leptin, immunoglobulins, single-chain
antibodies, complement components, serum albumin, cedar pollen
allergens, hypoxia-induced stress proteins, protein kinases,
proto-oncogene products, transcription factors and
virus-constitutive proteins.
11. A method for producing a foreign protein in a host cell,
comprising the steps of: culturing the cotransformant according to
claim 6 under suitable conditions so as to cause expression of
DsbA, DsbB, DsbC and DsbD, and the foreign protein; and purifying
the foreign protein from the host cell.
12. The method according to claim 11, wherein the suitable
conditions are those that cause expression of DsbA, DsbB, DsbC and
DsbD at levels wherein the foreign protein is in a solubilized
form.
13. The expression plasmid according to claim 5, further comprising
a polynucleotide encoding a foreign protein operably linked to a
promoter.
14. A transformant obtainable by introducing the expression plasmid
according to claim 13 into a host cell.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a DsbA/DsbB/DsbC/DsbD expression
plasmid. More particularly, the present invention relates to an
artificial operon comprising polynucleotides encoding each of DsbA,
DsbB, DsbC and DsbD, the operon being capable of expressing a
foreign protein in a soluble form while maintaining a normal
conformation, an expression plasmid carrying the operon, a
cotransformant harboring the expression plasmid and an expression
vector for a foreign protein as well as a method for producing a
foreign protein comprising culturing the cotransformant.
2. Discussion of the Related Art
Many of the eucaryote-derived proteins have disulfide bonds, and
they are not usually expected to have a natural tertiary structure
when expressed in the cytoplasm of E. coli under strong reductive
conditions. Therefore, in the production of such a protein, it is
considered to be effective to perform secretory expression into the
periplasm under oxidative conditions suitable for disulfide bond
formation. In addition to a strong possibility of expressing a
protein having its natural conformation, there can be expected
various advantages by expression via secretion, including a
possibility of expressing a protein which is toxic to cells; a
possibility of expressing a protein in which methionine is not
added at its N-terminal; and facilitation in purification owing to
a reduced amount of contaminant proteins. However, various reports
on attempts on secretion of heterologous proteins into the
periplasm of E. coli have been made, but not all heterologous
proteins can be expressed in the forms exhibiting their activities.
This is especially a problem in a case of a protein having a large
number of disulfide bonds.
On the other hand, in E. coli, there have been deduced the roles of
DsbA, DsbB, DsbC and DsbD which are the Dsb family proteins
involved in the formation of disulfide bonds by means of
biochemical tests and complementary tests using their respective
deletion strains [Bardwell, J. C., Mol. Microbiol. 14, 199-205
(1994); Sone, M. et al., J. Biol. Chem., 272, 10349-10352 (1997);
Rietsch, A. et al., Proc. Natl. Acad. Sci. USA, 93, 13048-13053
(1996)].
First, DsbA acts to form disulfide bonds in a nascent polypeptide
chain which has been transferred into the periplasm. The disulfide
bonds formed at this stage are not necessarily proper, and are then
corrected into proper disulfide bonds by means of cleavage of the
disulfide bonds followed by re-crosslinking by the action of DsbC.
Each of DsbA and DsbC has a thioredoxin-like active site motif
(Cys-X-X-Cys). In the Cys-X-X-Cys motif, 2 Cys residues are
considered to participate in the reaction. In the process of the
disulfide bond formation, the 2 Cys residues in the active center
of DsbA oxidize a substrate peptide chain, while they themselves
are reduced. Two Cys residues in the active center of DsbC are
cleaved as a result of the reduction of the disulfide bonds of the
substrate once formed, while they themselves are oxidized. Since a
reduced form of DsbA and an oxidized form of DsbC no longer have
catalytic activities, a factor for re-activating these DsbA and
DsbC is necessitated. Intracellular membrane protein DsbB
re-oxidizes DsbA, and intracellular membrane protein DsbD
re-reduces DsbC, respectively, by action of the thioredoxin-like
motifs existing in the periplasmic side.
For the purpose of improving secretion of a desired protein into
the periplasm, several attempts have been made to overexpress DsbA
or DsbC together with a desired protein, which could not so far be
said to be successful. For example, Knappik et al. disclose that
DsbA is required for the folding of an expressed product in the
secretion of an antibody fragment; however, there has yet remained
to be a problem that the efficiency of the folding does not change
even when overexpressed [Knappik, A. et al., Bio/Technol., 11,
77-83 (1993)]. In addition, Wunderlich and Glockschuber disclose
that the folding of an .alpha.-amylase/trypsin inhibitor is not
improved by the overexpression of DsbA, but increased to 14 times
in the presence of a reductive form of glutathione [Wunderlich, M.
and Glockschuber, R., J. Biol. Chem., 268, 24547-24550 (1993)].
Further, Wulfing and Pluckthum disclose that the overexpression of
DsbA exhibits some effects on the expression in soluble form of a T
cell receptor fragment in the periplasm; however, it is necessary
to overexpress simultaneously a heat shock sigma factor
.sigma..sup.32 in addition to DsbA [Wulfing, C. and Pluckthum, A.,
J. Mol. Biol., 242, 655-669 (1994)]. More recently, Joly et al.
have found that the overexpression of DsbA or DsbC serves to doubly
increase the expression level of an insulin-like growth factor I
(IGF-I) in the periplasm; however, there remains the disadvantage
that a soluble expression product is reduced contrary to
expectations [Joly, J. C. et al., Proc. Natl. Acad. Sci. USA, 95,
2773-2777 (1998)].
An object of the present invention is to provide an artificial
operon comprising polynucleotides encoding each of DsbA, DsbB, DsbC
and DsbD, the operon being capable of expressing a foreign protein
in a soluble form while maintaining a normal tertiary
structure.
In one embodiment, the present invention provides an expression
plasmid carrying the operon.
In another embodiment, the present invention provides a
cotransformant harboring the plasmid and an expression vector for a
foreign protein.
In still another embodiment, the present invention provides a
method for producing a foreign protein comprising culturing the
cotransformant.
These and other objects of the present invention will be apparent
from the following description.
SUMMARY OF THE INVENTION
One of the subject matter of the present invention is in the
findings that an accurate disulfide bond formation in the periplasm
can be surprisingly efficiently carried out, and a soluble
expression product can be further efficiently obtained when an
expression vector of the Dsb family proteins comprising a protein
(DsbA or DsbC) for forming or isomerizing disulfide bonds, as well
as a protein (DsbB or DsbD) which can control the reactivity of
DsbA or DsbC is constructed and the coexpression effects of these
proteins in the secretion of a foreign protein are studied.
In sum, the present invention pertains to the following: [1] an
artificial operon comprising polynucleotides encoding each of DsbA,
DsbB, DsbC and DsbD; [2] an expression plasmid carrying the
artificial operon according to item [1] above, usable for
expression of DsbA, DsbB, DsbC and DsbD; [3] a cotransformant
harboring the expression plasmid according to item [1] above and an
expression vector for a foreign protein; and [4] a method for
producing a foreign protein comprising culturing the cotransformant
according to item [3] above.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will become more fully understood from the
detailed description given hereinbelow and the accompanying
drawings which are given by way of illustration only, and thus, are
not limitative of the present invention, and wherein:
FIG. 1 is a schematic view showing pT7B/dsbA, pT7B/dsbB, pT7B/dsbC,
pT7B/dsbD and pT7B/dsbABCD;
FIG. 2 is a schematic view showing the construction of an
expression vector;
FIG. 3 is a schematic view showing an expression vector containing
pAR5/dsbA, pAR5/dsbB, pAR5/dsbC, pAR5/dsbD and pAR5/dsbABCD;
FIG. 4 shows analytic results on SDS-PAGE of the expressed Dsb
family proteins;
FIG. 5 shows changes in a total expression level of NGF-.beta. or
in a product level accumulated in each fraction, with respect to
the change in the arabinose concentration (W: whole cell; P:
periplasm);
FIG. 6 is a graph showing the growth of cells in the HRP
expression;
FIG. 7 shows changes in a total expression level of HRP or in a
product level accumulated in each fraction, with respect to the
change in the arabinose concentration;
FIG. 8 shows the changes of the OmpA-HRP levels after 0, 30, 85,
150 and 240 minutes from the addition of IPTG and the localization
(W: whole cell; P: periplasm soluble fraction; and S: spheroplast
fraction); and
FIG. 9 is a set of graphs showing the relative expression levels of
OmpA-HRP as determined from the results in FIG. 8, wherein the
upper drawing is a graph showing the results of the control, and
the lower drawing is a graph showing the results of
pAR5/dsbABCD.
DETAILED DESCRIPTION OF THE INVENTION
One of the significant features of the artificial operon of the
present invention resides in that the artificial operon comprises
genes encoding each of DsbA, DsbB, DsbC and DsbD, which are Dsb
family proteins. Since the artificial operon comprises the above
genes, there can be exhibited excellent effects that the disulfide
bonds of a foreign protein can be properly formed when coexpressed
with the foreign protein, whereby a soluble expression product can
be efficiently obtained.
In the present invention, the Dsb family proteins are ones involved
in the formation of disulfide bonds, and the Dsb family proteins
include DsbA, DsbB, DsbC and DsbD. It is considered that the DsbA
has a function for forming disulfide bonds in a nascent polypeptide
chain which has been transferred into the periplasm, and the DsbC
has a function for correcting the disulfide bonds formed already by
DsbA into proper disulfide bonds by means of cleavage of the
disulfide bonds followed by re-crosslinking. In addition, it is
considered that DsbB serves to re-oxidize DsbA, and DsbD serves to
re-reduce DsbC, respectively. The dsbA, dsbB, dsbC and dsbD genes
encoding each of the Dsb family proteins do not form any operons,
so that each of their expression is considered to be regulated
independently.
The Dsb family proteins described above include a protein derived
from E. coil, and their origins are not particularly limited as
long as they have the equivalent functions mentioned above.
Examples thereof include Salmonella typhimurium, Pseudomonas
aeruginosa, Haemophilus influenzae and the like. From the viewpoint
of expressing a foreign protein in a stabilized and soluble form in
E. coli, the Dsb family proteins derived from E. coli are
preferred.
The amino acid sequences of DsbA, DsbB, DsbC and DsbD are as shown
in SEQ ID NOs: 1, 3, 5 and 7 in the Sequence Listing, respectively,
and the nucleotide sequences of the genes encoding DsbA, DsbB, DsbC
and DsbD are as shown in SEQ ID NOs: 2, 4, 6 and 8 in the Sequence
Listing, respectively.
The amino acid sequences of DsbA, DsbB, DsbC and DsbD mentioned
above may further be a sequence in which a mutation such as
substitution, deletion, addition or insertion of one or more amino
acid residues is introduced into each of the amino acid sequences,
as long as the resulting polypeptide has an equivalent function as
above. In addition, two or more kinds of mutations may be
introduced in a sequence so long as the resulting polypeptide has
the equivalent functions as above. The above mutations can be
naturally-occurring or artificially-introduced mutations.
The nucleotide sequences of the dsbA, dsbB, dsbC and dsbD genes may
also be a sequence in which a mutation such as substitution,
deletion, addition or insertion of one or more bases is introduced
into each of the nucleotide sequences, as long as the nucleotide
sequence is encoded by the polypeptide having an equivalent
function as above. In addition, two or more kinds of mutations may
be introduced in a sequence so long as the nucleotide sequence has
the equivalent functions as above. In addition, the nucleotide
sequence may also be a nucleotide sequence consisting of genes
hybridizing to any of genes as shown in SEQ ID NOs: 2, 4, 6 and 8
in the Sequence Listing under stringent conditions, as long as the
nucleotide sequence is encoded by the polypeptide having an
equivalent function as above. Here, the hybridization conditions
are, for instance, those described in Molecular Cloning: A
Laboratory Manual, Second Ed. (Sambrook, J. et al., published by
Cold Spring Harbor Laboratory Press, New York, published 1989), and
the like.
The genes described above can be obtained by means of genetic
engineering techniques described in Molecular Cloning: A Laboratory
Manual, Second Ed. mentioned above, and the like.
Concretely, the gene described above can be obtained by means of,
for example, a screening method using a probe hybridizing to the
gene described above; a method comprising cleaving a fragment
containing a desired gene with an appropriate restriction enzyme,
and cloning the fragment; PCR method using a primer pair having a
sequence capable of amplifying each of the genes, and the like.
The method for obtaining the gene by PCR methods using a primer
pair having a sequence capable of amplifying each gene will be
described hereinbelow.
The primer used in PCR includes a primer having a sequence capable
of hybridizing under stringent conditions to the nucleotide
sequence of the genes described above or to a sequence
complementary thereto. In the primer described above, its
nucleotide sequence may have a restriction enzyme recognition site
in order to facilitate the operability. Primers for amplifying dsbA
gene are, for example, the primers as shown in SEQ ID NOs: 9 and 10
in the Sequence Listing. Primers for amplifying dsbB gene are, for
example, the primers as shown in SEQ ID NOs: 11 and 12 in the
Sequence Listing. Primers for amplifying dsbC gene are, for
example, the primers as shown in SEQ ID NOs: 13 and 14 in the
Sequence Listing. Primers for amplifying dsbD gene are, for
example, the primers as shown in SEQ ID NOs: 15 and 16 in the
Sequence Listing.
The template used for cloning by PCR methods includes, for
instance, pSK220 carrying dsbA and dsbB genes [Kamitani, S. et al.,
EMBO J., 11, 57-62 (1992)]; pSS51 carrying dsbA and dsbB genes
[Kishigami, S. and Ito, K., Genes Cells, I, 201-208 (1996)]; Kohara
Clone Nos. 468 and 648 carrying dsbC and dsbD genes [Kohara, Y. et
al., Cell, 50, 495-508 (1987)], and the like.
The composition for a reaction mixture, thermocycle for reaction,
and the like when carrying out the PCR method can be appropriately
set by observing the presence or absence of the resulting amplified
product. Concretely, in the amplification of, for instance, dsbA,
dsbB or dsbC gene, 25 cycles of reaction can be carried out,
wherein one cycle consists of 98.degree. C. for 5 seconds,
65.degree. C. for 2 seconds, and 74.degree. C. for 30 seconds, by
using 50 .mu.l of a reaction mixture having the composition of 50
pmol of a primer, 10 ng of template DNA, 1 U of KOD DNA polymerase
(manufactured by TOYOBO CO., LTD.), 0.2 mM dNTP, 6 mM
(NH.sub.4).sub.2 SO.sub.4, 1 mM KCl, 0.1% TRITON X-100, 0.001% BSA,
1 mM MgCl.sub.2 and 120 mM Tris-HCl (pH 8.0). In addition, in the
amplification of dsbD gene, 25 cycles of reaction can be carried
out after treatment at 94.degree. C. for 1 minute, wherein one
cycle consists of 98.degree. C. for 20 seconds and 68.degree. C.
for 3 minutes by using TAKARA LA TAQ.TM. (manufactured by Takara
Shuzo Co., Ltd.) in place of the KOD DNA polymerase in the above
PCR conditions, and further using a total volume of 50 .mu.l of a
reaction mixture [composition: 10 pmol of a primer, 2.5 ng of
template DNA, 2.5 U of TAKARA LA TAQ.TM., 0.4 mM dNTP, and
.times.10 TAKARA LA Buffer (pH 8.0)].
In the operon of the present invention, the order of the dsbA,
dsbB, dsbC and dsbD genes are not particularly limited as long as
the Dsb family proteins are expressed. An example includes a
polycistronic operon in which the genes are arranged in tandem in
the order of dsbA-dsbB-dsbC-dsbD, and the like. Incidentally, the
nucleotide sequence of the polycistronic operon in which genes are
arranged in tandem in the order of dsbA-dsbB-dsbC-dsbD mentioned
above is as shown in SEQ ID NO: 17 in the Sequence Listing.
In addition, it is preferable that each of the dsbA, dsbB, dsbC and
dsbD genes has a ribosomal binding site (SD sequence) in the
upstream of its respective structural gene, and it is more
preferable that each of these genes has a ribosomal binding site 7
to 10 bp upstream of its respective structural gene.
In the operon of the present invention, the genes may be present
under the control of a promoter. From the viewpoint of regulation
of the expression level of the Dsb family proteins, it is
preferable that the promoter for controlling the transcription of
the above-described operon which is present under the control of a
promoter is an inducible promoter. Examples of the inducible
promoter include, for instance, lac, tac, trc, trp, ara, Pzt-1,
P.sub.L and T7. The lac, tac and trc promoters can be induced by
using isopropyl-.beta.-D-thio-galactopyranoside (IPTG); the trp,
ara and Pzt-1 promoters can be induced by using 3-indoleacrylic
acid (IAA), L-arabinose and tetracycline, respectively. The P.sub.L
promoter can be induced at a high temperature (42.degree. C.). Also
usable is T7 promoter, which is specifically and strongly
transcribed by T7 RNA polymerase. In a case where the T7 promoter
is used, the T7 promoter can be induced with IPTG by using as a
host E. coli strain harboring a lysogenized .lambda. phage carrying
the T7 RNA polymerase gene located downstream of the lac promoter.
Among the promoters, lac, tac, trc, trp, ara, Pzt-1 and T7 are
preferable from the viewpoint of facilitation in induction
operability. The above promoter is contained in a known vector, and
can be used by appropriately cleaving from the vector with a
restriction enzyme, and the like.
In the operon of the present invention, the Dsb family proteins can
be expressed more stably when the operon carries a terminator such
as rrnBT1T2. These terminators are contained in a known vector, and
can be used by appropriately cleaving from the vector with a
restriction enzyme, or the like.
One of the significant features of the expression plasmid of the
present invention resides in that DsbA, DsbB, DsbC and DsbD can be
expressed by the expression plasmid, and that the expression
plasmid carries the operon described above.
As described above, it is preferable that the expression plasmid of
the present invention expresses the Dsb family proteins of the
present invention, namely DsbA, DsbB, DsbC and DsbD under the
control of an inducible promoter.
In addition, when the expression plasmid of the present invention
is introduced into a host, a plasmid such as the same plasmid and
the operon described above and a gene encoding a desired foreign
protein may be used, or separate plasmids for carrying either one
of the operon or the gene encoding a foreign protein (hereinafter
referred to as coexpression plasmid) may also be used. Among them,
the coexpression plasmids are preferred from the viewpoints of not
necessitating to prepare a plasmid for each foreign protein as well
as the stability of the plasmid in a host. The term "foreign
protein" used herein refers to a desired protein except for DsbA,
DsbB, DsbC and DsbD.
In order to optimize the expression level and the timing of
expression of the Dsb family proteins described above without
lowering the expression level of a foreign protein, it is more
advantageous to independently control the expression of the Dsb
family proteins from the expression of the desired protein. An
inducible promoter used for expression of the Dsb family protein is
preferably one different from that used in the expression of the
desired protein.
When a coexpression plasmid is used as the expression plasmid
described above, any plasmid can be used as long as the plasmid has
a replicon compatible with an expression vector for a desired
protein in E. coli used as a host. For example, when a vector
having ColE1 replicon such as pBR322 is used as an expression
vector for a desired protein, p15A replicon present in pACYC vector
can be used for a plasmid used for expression of the Dsb family
proteins of the present invention.
Concrete examples of the expression plasmid of the present
invention include a coexpression plasmid pAR5/dsbABCD. This
pAR5/dsbABCD is, as shown in FIG. 3, a plasmid resulting from
sequential insertions of dsbA, dsbB, dsbC and dsbD genes at the
multicloning site of plasmid pAR5, wherein pAR5 carries a
chloramphenicol-resistant gene and ara promoter capable of inducing
expression with arabinose, which are derived from pAR3, a
derivative of pACYC184 vector [Perez et al., Gene, 158, 141-142
(1995)], as well as the multicloning site and rrnBT1T2 terminator
derived from pTrc99A (manufactured by Pharmacia) downstream of the
above ara promoter. The pAR5/dsbABCD can induce the expression of
the Dsb family proteins described above by adding arabinose. The
pAR5/dsbABCD can also contribute to a proper disulfide bond
formation in a foreign protein in the co-presence of other plasmid
carrying a gene encoding the foreign protein, whereby producing a
soluble expression product at a high efficiency.
The above-described plasmid can be constructed by a method, for
example, described in Molecular Cloning: A Laboratory Manual, 2nd
Ed. mentioned above.
The plasmid of the present invention may further contain a
selection marker gene as occasion demands in order to facilitate
selection upon transformation. Examples of such selection marker
genes include ampicillin resistance (Amp.sup.r) genes, kanamycin
resistance (Km.sup.r) genes, chloramphenicol resistance (Cm.sup.r)
genes, and the like. It is desired that in the coexpression
plasmid, the selection marker gene is different from the selection
marker gene contained in the expression vector for a foreign
protein.
One of the significant features of the cotransformant of the
present invention resides in that the cotransformant harbors the
expression plasmid described above (coexpression plasmid) as well
as an expression vector for a foreign protein.
The above cotransformant can be obtained by cotransforming an
expression plasmid (the coexpression plasmid) typically exemplified
by the pAR5/dsbABCD described above together with an expression
vector for a foreign protein carrying a gene encoding the foreign
protein.
The expression vector for a foreign gene used in the cotransformant
described above is not particularly limited, and it may be a vector
capable of expressing a desired foreign protein in the cytoplasm of
a cell or capable of secreting a desired foreign protein into the
periplasm of a cell, wherein the vector exhibits compatibility with
the expression plasmid described above. Particularly preferable is
a vector in which the expression of a desired foreign protein can
be induced under the control of an inducible promoter. The
inducible promoter includes promoters similar to those described
above. The Dsb family proteins and a desired protein can separately
be induced for expression by selecting a promoter other than the
promoter used in the induction for expression of the Dsb family
proteins in the present invention.
In addition, the expression vector for a foreign gene may also
comprise a selective marker gene as occasion demands. The above
selective marker gene includes those described above, and double
selection of the cotransformant can be achieved by using a
selective marker gene other than that contained in the expression
plasmid (coexpression plasmid) of the present invention.
The expression vector for a foreign gene described above is
preferably a vector capable of secreting into the periplasm of a
cell, from the viewpoint of forming proper disulfide bonds in the
resulting foreign protein. Examples of the vector include a vector
carrying a gene encoding a polypeptide formed by adding a signal
peptide of OmpA, OmpT, MalE, .beta.-lactamase, or the like to a
desired foreign protein. The above vector can be obtained, for
example, by adding a polynucleotide encoding the signal peptide
mentioned above by means of genetic engineering technique to a
position on a gene corresponding to the N-terminal of a desired
foreign protein, and incorporating the resulting gene into a known
vector.
In addition, the expression vector for a foreign gene of the
present invention may also contain a sequence which enables to
carry out a technique for facilitating purification of a desired
foreign protein, typically exemplified by, for instance, expression
as a fusion protein with a protein such as .beta.-galactosidase,
glutathione-S-transferase and maltose-binding protein; expression
as a protein having an added histidine tag, and the like, so long
as the objects of the present invention are not hindered.
Concrete examples of host E. coli strains usable in the present
invention include generally used strains, such as HB101, JM109,
MC4100, MG1655 and W3110; and various mutants, including protease
mutants, such as degP mutants, ompT mutants, tsp mutants, lon
mutants, clpPX mutants, hslV/U mutants, lon-cLpPX double mutants
and lon-clpPX-hslV/U triple mutants; plsX mutants; rpoH deletion
mutants; rpoH missense mutants, and the like.
In the present invention, concrete examples of protease mutants
include degP mutants, ompT mutants, tsp mutants, lon mutants,
lon-clpPX double mutants and lon-clpPX-hslV/U triple mutants are
preferable from the viewpoint of more stably expressing a foreign
protein.
Here, a preferable lon-clpPX double mutant is E. coli strain KY2783
derived from E. coli strain W3110, prepared by introducing double
deletion mutations in the lon and clpPX genes [named and identified
as E. coli KY2783 and has been deposited under accession number
FERM BP-6244 with the National Institute of Bioscience and
Human-Technology, Agency of Industrial Science and Technology,
Ministry of International Trade and Industry, of which the address
is 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan;
date of original deposit: Feb. 3, 1998].
Also, the term "lon-clpPX-hslV/U triple mutant" refers to a mutant
prepared by further introducing a mutation of the hslV/U gene,
which encodes HslV/U protease, in the above-described lon-clpPX
double mutant. A preferable lon-clpPX-hslV/U triple mutant is E.
coli strain KY2893 derived from E. coli strain W3110, prepared by
introducing triple deletion mutations in the lon, clpPX and hslV/U
genes [named and identified as E. coli KY2893 and has been
deposited under accession number FERM BP-6243 with the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry, of which the address is 1-3, Higashi 1-chome,
Tsukuba-shi, Ibaraki-ken, 305-8566, Japan; date of original
deposit: Feb. 3, 1998].
In the present invention, the foreign protein to be expressed may
be any protein, as long as it is a foreign protein that is
expressed in unstabilized form and/or insoluble form in a host,
particularly in E. coli. Such foreign proteins include interferons,
interleukins, interleukin receptors, interleukin receptor
antagonists, granulocyte colony-stimulating factors, granulocyte
macrophage colony-stimulating factors, macrophage
colony-stimulating factors, erythropoietin, thrombopoietin,
leukemia inhibitors, stem cell growth factors, tumor necrosis
factors, growth hormones, proinsulin, insulin-like growth factors,
fibroblast growth factors, platelet-derived growth factors,
transforming growth factors, hepatocyte growth factors, bone
morphogenetic factors, nerve growth factors, ciliary neurotrophic
factors, brain-derived neurotrophic factors, glia cell line-derived
neurotrophic factors, neurotrophin, angiogenesis inhibitors,
prourokinase, tissue plasminogen activators, blood coagulation
factors, protein C, glucocerebrosidase, superoxide dismutase,
renin, lysozyme, P450, prochymosin, trypsin inhibitors, elastase
inhibitors, lipocortin, leptin, immunoglobulins, single-chain
antibodies, complement components, serum albumin, cedar pollen
allergens, hypoxia-induced stress proteins, protein kinases,
proto-oncogene products, transcription factors and
virus-constitutive proteins.
As a method for introducing the expression plasmid of the present
invention into E. coli together with an expression vector for a
foreign protein, there can be employed conventional methods such as
the calcium chloride method, rubidium chloride method,
electroporation method and other conventional methods. The
cotransformant can be screened by using chemicals in accordance
with the selection marker genes. The expression of the foreign
protein can, for example, be confirmed by such means as Western
blotting analysis.
One of the significant features of the method for producing a
foreign protein of the present invention resides in culturing the
cotransformant described above. The production method can, for
example, be carried out by a process comprising culturing a
cotransformant under induction conditions for the Dsb family
proteins suitable for stabilization and/or solubilization of a
desired foreign protein to allow expression of the Dsb family
proteins and the foreign protein; thereafter collecting the cells;
disrupting the collected cells; and isolating and purifying the
foreign protein in accordance with a purification method suitable
for the foreign protein.
The induction conditions described above vary with inducible
promoters used in the expression plasmid of the present invention
and the expression vector for a foreign protein, and the conditions
may be such that the expression levels of DsbA, DsbB, DsbC and DsbD
are at levels suitable for the foreign protein to be solubilized.
For example, the induction conditions can be determined as
follows.
First, an inducer for the promoter described above is added with
varying its concentrations and addition timings. The cells
expressing a foreign protein are collected, and the collected cells
are disrupted and extracted to obtain cell extracts. Each of the
resulting extracts is subjected to, for instance, SDS-PAGE, and the
bands ascribed to the proteins in the gel are visualized by
Coomassie brilliant blue- or silver-staining. Among the visualized
bands, the concentration of the band ascribed to the foreign
protein is examined by, for instance, a densitometry, whereby
finding appropriate induction conditions.
Since the culture conditions of the cotransformant vary with the
microorganism used as a host, they are not particularly limited.
The optimum conditions can be determined by examining the
expression level of a foreign protein expressed under each of
culturing conditions by setting various culture timing and culture
temperatures, in a manner similar to the case of the determination
of the induction conditions described above.
The foreign protein can be isolated and purified by any known
protein purification methods, including salting-out, ion exchange
chromatography, hydrophobic chromatography, affinity chromatography
and gel filtration chromatography.
EXAMPLES
The present invention will be further described hereinbelow by
means of the following Examples, and the present invention is by no
means limited to these Examples.
Example 1
Cloning of Genes Encoding Dsb Family Proteins
Each of genes dsbA, dsbB, dsbC and dsbD, each of which is a
structural gene respectively encoding DsbA, DsbB, DsbC and DsbD,
which are Dsb family proteins, was cloned by PCR method. As the
primers, those which were designed so that a ribosomal binding site
should be located at 7 to 10 bp upstream in each of the structural
genes were used. There were used as the primers for amplifying the
dsbA gene, the primers as shown in SEQ ID NOs: 9 and 10 in the
Sequence Listing; as the primers for amplifying the dsbB gene, the
primers as shown in SEQ ID NOs: 11 and 12 in the Sequence Listing;
as the primers for amplifying the dsbC gene, the primers as shown
in SEQ ID NOs: 13 and 14 in the Sequence Listing; and as the
primers for amplifying the dsbD gene, the primers as shown in SEQ
ID NOs: 15 and 16 in the Sequence Listing. Incidentally, the
primers were so designed to have restriction enzyme recognition
sites as given below in the nucleotide sequences. Primer (SEQ ID
NO: 9): SacI; Primer (SEQ ID NO: 10): AvaI; Primer (SEQ ID NO: 11):
AvaI; Primer (SEQ ID NO: 12): NdeI; Primer (SEQ ID NO: 13): NdeI;
Primer (SEQ ID NO: 14): SalI; Primer (SEQ ID NO: 15): SalI; and
Primer (SEQ ID NO: 16): SphI.
As templates, there were used pSK carrying dsbA and dsbB genes
[Kamitani, S. et al., EMBO J., 11, 57-62 (1992)]; pSS51 [Kishigami,
S. and Ito, K., Genes Cells, I, 201-208 (1996)]; and Kohara Clone
Nos. 468 and 648 carrying dsbC and dsbD genes [Kohara, Y. et al.,
Cell, 50, 495-508 (1987)], which were made available by Dr.
Yoshinori Akiyama, The Institute of Virus Research, Kyoto
University.
The PCR conditions are given hereinbelow.
There was obtained 50 .mu.l of a reaction mixture having the
composition of 50 pmol of each of primers, 10 ng of template DNA, 1
U of KOD DNA polymerase (manufactured by TOYOBO CO., LTD.), 0.2 mM
dNTP, 6 mM (NH.sub.4).sub.2 SO.sub.4, 1 mM KCl, 0.1% TRITON X-100,
0.001% BSA, 1 mM MgCl.sub.2 and 120 mM Tris-HCl (pH 8.0). The
resulting reaction mixture was set in GENEAMP.TM. PCR System 2400
(manufactured by Perkin-Elmer), and 25 cycles of reaction were
carried out, wherein one cycle consisted of 98.degree. C. for 5
seconds, 65.degree. C. for 2 seconds, and 74.degree. C. for 30
seconds.
As a result of carrying out PCR under the conditions described
above, when pSK220 and pSS51 carrying dsbA and dsbB genes were used
as templates, there was found specific amplification of about 0.6
kb and about 0.5 kb fragments which have been considered to be
corresponding to dsbA and dsbb genes, respectively. On the other
hand, when Kohara Clone No. 468 carrying dsbC gene was used as a
template, there was found specific amplification of a fragment of
about 0.7 kb which has been considered to be corresponding to dsbC
gene. Alternatively, when Kohara Clone No. 648 carrying dsbD gene
was used as a template, there was found to be no amplification of
the fragment corresponding to dsbD gene.
Therefore, in order to clone the dsbD gene, after treatment at
94.degree. C. for 1 minute, 25 cycles of reaction can be carried
out wherein one cycle consisted of 98.degree. C. for 20 seconds and
68.degree. C. for 3 minutes by using TaKaRa LA Taq.TM.
(manufactured by Takara Shuzo Co., Ltd.) in place of the KOD DNA
polymerase in the above PCR conditions, and further using a total
volume of 50 .mu.l of a reaction mixture [composition: 10 pmol of
each of primers, 2.5 ng of template DNA, 2.5 U of TaKaRa LA
Taq.TM., 0.4 mM dNTPs, and .times.10 TaKaRa LA Buffer (pH 8.0)]. As
a template, Kohara Clone No. 648 was used. As a result, there was
found an amplification of about 1.5 kb fragment, which is
considered to correspond to the dsbD gene.
The nucleotide sequence of the amplified fragment thus obtained was
determined, and as a result, it was elucidated that each of genes
dsbA, dsbB, dsbC and dsbD was obtained. The nucleotide sequences of
dsbA, dsbB, dsbC and dsbD are as shown in SEQ ID NOs: 2, 4, 6 and 8
in the Sequence Listing, respectively.
Subsequently, each of the amplified fragments obtained as described
above was ligated to the multicloning site of pT7Blue(R)
(manufactured by Novagen), whereby constructing each of the
plasmids resulting from singly ligating any one of dsbA, dsbB, dsbC
or dsbD to pT7Blue(R) (each plasmid being referred to as pT7B/dsbA,
pT7B/dsbB, pT7B/dsbC and pT7B/dsbD, respectively); as well as a
plasmid resulting from ligating to pT7Blue(R) an operon (SEQ ID NO:
17) obtained by tandemly connecting dsbA, dsbB, dsbC and dsbD
(hereinafter referred to as "pT7B/dsbABCD"). Each of the plasmids
is shown in FIG. 1.
Example 2
Construction of Expression Vector
It is considered that the 4 genes (dsbA, dsbB, dsbC and dsbD)
encoding the Dsb family proteins do not form an operon, and
expression of each gene is regulated independently. Accordingly,
there was constructed an expression vector in which the 4 genes
form a polycistronic operon, and expression of these genes is
inducible by adding arabinose. FIG. 2 shows the strategy of
construction.
In order that the structural genes for the Dsb family proteins can
be expressed independently from a structural gene for a model
protein, the synthetic DNA (SEQ ID NO: 18) having a recognition
sequence of various restriction enzymes was ligated to the
PstI-HindIII site of pAR3, wherein pAR3 [chloramphenicol-resistant
and capable of inducing and expressing with arabinose] was derived
from pACYC184, which was constructed by Perez et al. [see Gene,
158, 141-142 (1995)], to give pAR4 having a multicloning site.
Subsequently, a fragment comprising rrnBT1T2 terminator was cut out
by treating pTrc99A (manufactured by Pharmacia) with PvuI,
blunt-ending the resulting fragment with Mung Bean Nuclease, and
thereafter treating the blunt-ended fragment with SacI. The
resulting fragment was ligated to the SacI-NruI sites of the
above-described pAR4, to give pAR5. The pAR5 mentioned above is
capable of expressing in the coexistence of a plasmid carrying ori
of pBR322, so that the expression level of an expression product
derived from a foreign gene inserted into the multicloning site can
be regulated by adding arabinose.
Example 3
Construction of Expression Plasmids for Dsb Family Proteins and
Confirmation of Expression Thereof
A SacI-HindIII fragment was cut out from each of plasmids
pT7Blue(R)/dsbA, pT7Blue(R)/dsbB, pT7Blue(R)/dsbC, pT7Blue(R)/dsbD
and pT7Blue(R)/dsbABCD each obtained in Example 1, and then the
fragment was inserted into the SacI-HindIII sites in the
multicloning site of pAR5 obtained in Example 2. FIG. 3 shows the
resulting expression plasmids in which any one of DsbA, DsbB, DsbC
and DsbD is ligated respectively thereto (referred to as pAR5/dsbA,
pAR5/dsbB, pAR5/dsbC and pAR5/dsbD, respectively), as well as the
expression plasmid in which these 4 inserts are tandemly connected
to form a polycistronic operon (pAR5/dsbABCD).
Expression of the Dsb family proteins was attempted by transforming
E. coli JM109 with each of the plasmids obtained as described
above. Competent cells were obtained from E. coli JM109 by the
PEG-DMSO method and transformed with 1.0.times.10.sup.-2 .mu.g of
any one of the above expression plasmids pAR5/dsbA, pAR5/dsbB,
pAR5/dsbC, pAR5/dsbD and pAR5/dsbABCD. Screening of the
transformants was carried out by using their resistivity to
chloramphenicol as an index.
Subsequently, each of the resulting transformants was cultured at
37.degree. C. in 5 ml of L medium containing 34 .mu.g/ml
chloramphenicol. Arabinose was added so as to have the final
concentrations of 0, 200 and 2000 .mu.g/ml, respectively, when
Klett units reached around 20. Two hours later, each culture was
sampled, and the cells contained in the resulting culture were
harvested. The cells were subjected to precipitation treatment with
TCA, to give whole cell protein. The whole cell protein obtained as
above was subjected to SDS-PAGE analysis. The results are shown in
FIG. 4.
As shown in FIG. 4, it was confirmed that when pAR5/dsbC and
pAR5/dSbABCD were used, bands with a molecular weight of about
24000 which were deduced to be corresponding to DsbC, were
increased significantly depending on the arabinose
concentration.
Although no bands corresponding to the other Dsb products could be
detected, the expression of each of the Dsb family proteins was
confirmed by examining the presence or absence of the
complementation of each of deletion mutation of dsbA, dsbB and
dsbD. As a result, it could be confirmed that the function for each
of the Dsb family proteins was complemented in each Dsb-deletion
mutant.
Example 4
Construction of Expression Plasmids pTrc-OmpA and pTrc-OmpT for
Secretion of Foreign Protein
A plasmid resulting from insertion of a synthetic oligonucleotide
(5'-terminal: blunt end; 3-terminal: NaeI and EcoRI sites) encoding
the signal peptide of OmpA or OmpT as shown below into the
NcoI(blunted with Mung Bean Nuclease)-EcoRI sites of the expression
plasmid pTrc99A for E. coli was named pTrc-OmpA or pTrc-OmpT.
Oligonucleotide encoding OmpA signal sequence [Met Lys Lys Thr Ala
Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala Asn Ala
(SEQ ID NO: 19)]:
Oligonucleotide encoding OmpT signal sequence [Met Arg Ala Lys Leu
Leu Gly Ile Val Leu Thr Thr Pro Ile Ala Ile Ser Ser Phe Ala (SEQ ID
NO: 21)]:
Example 5
Construction of Secretion Plasmid for NGF-.beta.
An EcoRI-BamHI fragment of cDNA (manufactured by R&D Systems)
encoding an amino acid sequence in which N-terminal signal sequence
portion of human nerve growth factor-.beta. (NGF-.beta.) was
deleted was inserted into the EcoRI-BamHI sites of pTrc-OmpT
obtained in Example 4. Subsequently, the following synthetic
oligonucleotide linker (blunt end-EcoRI site) corresponding to the
N-terminal portion of NGF was treated with polynucleotide kinase
and thereafter inserted to the NaeI-EcoRI site of the resulting
plasmid, to give a secretion plasmid pTrc-OmpT/NGF for
NGF-.beta..
NGF Linker:
Example 6
Construction of Secretion Plasmid for HRP
The region corresponding to the structural gene was amplified by
PCR method using horseradish peroxidase (HRP) cDNA (manufactured by
R&D Systems) as a template. The resulting fragment was treated
with BamHI and then with polynucleotide kinase, and thereafter the
treated fragment was inserted into the NaeI-EcoRI sites of
pTrc-OmpA, to give a secretion plasmid pTrc-OmpA/HRP for HRP. The
primers used in the PCR method are as shown below. Primer:
Example 7
Enhancement Effect of DsbABCD on Expression of Foreign Protein in
Periplasm
The effects of DsbABCD (influence on growth of cells, changes in
the expression level and localization of the product) were examined
in the cells cotransformed with the NGF-.beta. secretion plasmid or
the HRP secretion plasmid obtained in Example 5 or 6 and
pAR5/dsbABCD. For each of cells grown at 37.degree. C. in the L
medium, DsbABCD was induced by adding arabinose (0 to 2000
.mu.g/ml), and NGF-.beta. or HRP was each induced by adding IPTG
(50 .mu.M). Each of accumulated proteins in cells were subjected to
SDS-PAGE analysis and Western blotting method. The whole cell
extract was obtained in the same manner as in Example 3. In
addition, the periplasm soluble fraction was obtained by a method
of treating the cells with lysozyme in the presence of isotonic
sucrose [Koshland, D. and Botstein, D., Cell, 20, 749-760
(1980)].
(1) Effects on NGF Expression
When pTrc-OmpT/NGF and vector pACYC184 without inserts were
coexistent, the growth of the cells was not found to be inhibited
even when the arabinose concentration increased up to 200 .mu.g/ml,
but the growth of the cells tended to be inhibited at the arabinose
concentration of 2000 .mu.g/ml. On the other hand, when
pTrc-OmpT/NGF and pAR5/dsbABCD were coexistent, the growth of the
cells was not inhibited at all, and their growth rate increased
about 10% at its maximum as the arabinose concentration
increased.
Subsequently, in order to study the expression level of NGF-.beta.
in the whole cell, or the amount of NGF-.beta. accumulated in the
periplasm soluble fraction, with varying the arabinose
concentration, 80 .mu.l of sample corresponding to a culture medium
with Klett units 80 of the whole cell and the periplasm soluble
fraction was subjected to SDS-PAGE analysis. The results of
SDS-PAGE analysis are shown in FIG. 5.
As shown in FIG. 5, in the control where pTrc-Ompt/NGF and vector
pACYC184 were coexistent, both the expression level of
OmpT-NGF-.beta. in the whole cell and in the periplasm soluble
fraction were hardly changed even when the arabinose concentration
was changed from 0 to 200 .mu.g/ml, and the expression level of the
product OmpT-NGF-.beta. was significantly lowered at the arabinose
concentration of 2000 .mu.g/ml. A total expression level of
OmpT-NGF-.beta. in the coexistence of pTrc-OmpT/NGF and
pAR5/dsbABCD was hardly changed (approximately 1 to 2 mg/l culture)
even when the arabinose concentration was changed from 0 to 2000
.mu.g/ml. The expression level of OmpT-NGF-.beta. in the periplasm
soluble fraction tended to be increased as the arabinose
concentration was raised, so that almost all of the expressed
OmpT-NGF-.beta. were detected in the periplasm soluble
fraction.
(2) Effect on HRP Expression
The growth of strains measured by Klett units in expression of HRP
examined are shown in FIG. 6. In addition, in order to examine the
expression level of HRP in the whole cell or the amount of HRP
accumulated in the periplasm soluble fraction with varying
concentrations of arabinose, a sample containing 60 .mu.l culture
with Klett units 80 of each of the whole cell or the periplasm
soluble fraction was subjected to SDS-PAGE analysis. The results of
SDS-PAGE analysis are shown in FIG. 7.
As shown in FIG. 6, in the case where pTrc-OmpA/HRP and
pAR5/dsbABCD were coexistent, when arabinose was not added, growth
of the cells stopped about 2 hours after adding IPTG, thereby
showing significant inhibition of growth of the cells, and when
arabinose was added (final concentration: 200 .mu.g/ml), the
inhibition of growth of the cells was eliminated, and the growth of
the cells did not stop even at 4 hours after adding IPTG. When
pTrc-OmpA/HRP and vector pAR3 without inserts were coexistent, the
inhibition of growth of the cells was not ameliorated at all. These
results suggest that the elimination of growth inhibition when
OmpA-HRP is expressed depends on expression of DsbABCD.
Also, as shown in FIG. 7, when pTrc-OmpA/HRP and pAR5/dsbABCD were
coexistent, the expression level of OmpA-HRP in the whole cell was
about 2 to 3 times that of the case where pTrc-OmpA/HRP and vector
pAR3 without inserts were coexistent. When pTrc-OmpA/HRP and
pAR5/dsbABCD were coexistent, the expression level of OmpA-HRP in
the periplasm soluble fraction was also increased in accordance
with the arabinose concentration.
Example 8
Changes in Expression Level and Localization of HRP Product in
Prolonged Induction
In order to examine the time course of the expression level and
localization of the expression products, Omp-HRP and DsbABCD were
induced and coexpressed by adding IPTG in the same manner as in
Example 6 in the coexistence of pTrc-OmpA/HRP and pAR5/dsbABCD. As
the control, the expression system in which pTrc-OmpA/HRP and
vector pAR3 without inserts were coexistent was used. After adding
IPTG, the culture was sampled at 0, 30, 85, 150, and 240 minutes
thereafter in order to examine the changes in the expression level
and the localization (the whole cell, the periplasm soluble
fraction, or the spheroplast fraction) of OmpA-HRP contained in the
culture. The whole cell and the periplasm soluble fraction were
obtained in the same manner as in Examples 3 and 7. In addition,
the spheroplast fraction was obtained as a fraction remaining after
extraction of the periplasm soluble fraction by the lysozyme
method. A sample containing the whole cell, the periplasm soluble
fraction or the spheroplast fraction, which corresponded to 60
.mu.l of culture with Klett units 80, was subjected to SDS-PAGE
analysis and Western blotting to determine a relative expression
level of OmpA-HRP. The results are shown in FIGS. 8 and 9. In FIG.
8, W is whole cell; P is a periplasm soluble fraction; and S is a
spheroplast fraction.
It is shown from the results in FIG. 8 that in the system where
OmpA-HRP and DsbABCD are coexpressed, the accumulation of OmpA-HRP
is initiated about 30 minutes after adding IPTG, and thereafter
reaches the maximum about 150 minutes after adding IPTG. In this
expression level, HRP could be also confirmed by CBB staining.
Also, it is shown in the results in FIG. 9 that the expression
level of HRP in the periplasm soluble fraction, relative to the
total amounts of HRP expressed, was about 10% after 85 minutes, but
surprisingly increased up to about 60% after 240 minutes. By
contrast, it is shown in the results in FIG. 9 that in the system
in which pTrc-OmpA/HRP and vector pAR3 without inserts were
coexistent, the accumulation of OmpA-HRP is at last initiated 150
minutes after adding IPTG (transition phase from the logarithmic
growth phase to the stationary phase), and the accumulation thereof
in the periplasm hardly occurs, showing accumulation of about 3% of
whole expression level. Further, it is clear from the results in
FIG. 6 that growth inhibition occurs along with expression of
OmpA-HRP, whereby stopping the growth.
According to the present invention, there can be exhibited an
excellent effect that a soluble expression product can be
efficiently obtained because the formation of accurate disulfide
bonds in the periplasm can be efficiently performed.
Equivalents
Those skilled in the art will recognize, or be able to ascertain
using simple routine experimentation, many equivalents to the
specific embodiments of the invention described in the present
specification. Such equivalents are intended to be encompassed in
the scope of the following claims.
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 25 <210>
SEQ ID NO 1 <211> LENGTH: 208 <212> TYPE: PRT
<213> ORGANISM: Escherichia coli <400> SEQUENCE: 1 Met
Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe 5 10 15 Ser
Ala Ser Ala Ala Gln Tyr Glu Asp Gly Lys Gln Tyr Thr Thr 20 25 30
Leu Glu Lys Pro Val Ala Gly Ala Pro Gln Val Leu Glu Phe Phe 35 40
45 Ser Phe Phe Cys Pro His Cys Tyr Gln Phe Glu Glu Val Leu His 50
55 60 Ile Ser Asp Asn Val Lys Lys Lys Leu Pro Glu Gly Val Lys Met
65 70 75 Thr Lys Tyr His Val Asn Phe Met Gly Gly Asp Leu Gly Lys
Glu 80 85 90 Leu Thr Gln Ala Trp Ala Val Ala Met Ala Leu Gly Val
Glu Asp 95 100 105 Lys Val Thr Val Pro Leu Phe Glu Gly Val Gln Lys
Thr Gln Thr 110 115 120 Ile Arg Ser Ala Ser Asp Ile Arg Asp Val Phe
Ile Asn Ala Gly 125 130 135 Ile Lys Gly Glu Glu Tyr Asp Ala Ala Trp
Asn Ser Phe Val Val 140 145 150 Lys Ser Leu Val Ala Gln Gln Glu Lys
Ala Ala Ala Asp Val Gln 155 160 165 Leu Arg Gly Val Pro Ala Met Phe
Val Asn Gly Lys Tyr Gln Leu 170 175 180 Asn Pro Gln Gly Met Asp Thr
Ser Asn Met Asp Val Phe Val Gln 185 190 195 Gln Tyr Ala Asp Thr Val
Lys Tyr Leu Ser Glu Lys Lys 200 205 <210> SEQ ID NO 2
<211> LENGTH: 647 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 2 atcggagaga gtagatcatg
aaaaagattt ggctggcgct ggctggttta gttttagcgt 60 ttagcgcatc
ggcggcgcag tatgaagatg gtaaacagta cactaccctg gaaaaaccag 120
ttgctggcgc gccgcaagtg ctggagtttt tctctttctt ctgcccgcac tgctatcagt
180 ttgaagaagt tctgcatatt tctgataacg tgaagaaaaa actgccggaa
ggcgtgaaga 240 tgactaaata ccacgtcaac ttcatggggg gtgacctggg
caaagagctg actcaggcat 300 gggctgtggc gatggcgctg ggcgtggaag
acaaagtcac agttccgctg tttgaaggcg 360 tacaaaaaac ccagaccatt
cgttcagcat ctgatatccg cgatgtattt atcaacgcag 420 gtattaaagg
tgaagagtac gacgcggcgt ggaacagctt cgtggtgaaa tctctggtcg 480
ctcagcagga aaaagctgca gctgacgtgc aattgcgtgg tgttccggcg atgtttgtta
540 acggtaaata tcagctgaat ccgcagggta tggataccag caatatggat
gtttttgttc 600 agcagtatgc tgatactgtg aaatatctgt ccgagaaaaa ataataa
647 <210> SEQ ID NO 3 <211> LENGTH: 176 <212>
TYPE: PRT <213> ORGANISM: Escherichia coli <400>
SEQUENCE: 3 Met Leu Arg Phe Leu Asn Gln Cys Ser Gln Gly Arg Gly Ala
Trp 5 10 15 Leu Leu Met Ala Phe Thr Ala Leu Ala Leu Glu Leu Thr Ala
Leu 20 25 30 Trp Phe Gln His Val Met Leu Leu Lys Pro Cys Val Leu
Cys Ile 35 40 45 Tyr Glu Arg Cys Ala Leu Phe Gly Val Leu Gly Ala
Ala Leu Ile 50 55 60 Gly Ala Ile Ala Pro Lys Thr Pro Leu Arg Tyr
Val Ala Met Val 65 70 75 Ile Trp Leu Tyr Ser Ala Phe Arg Gly Val
Gln Leu Thr Tyr Glu 80 85 90 His Thr Met Leu Gln Leu Tyr Pro Ser
Pro Phe Ala Thr Cys Asp 95 100 105 Phe Met Val Arg Phe Pro Glu Trp
Leu Pro Leu Asp Lys Trp Val 110 115 120 Pro Gln Val Phe Val Ala Ser
Gly Asp Cys Ala Glu Arg Gln Trp 125 130 135 Asp Phe Leu Gly Leu Glu
Met Pro Gln Trp Leu Leu Gly Ile Phe 140 145 150 Ile Ala Tyr Leu Ile
Val Ala Val Leu Val Val Ile Ser Gln Pro 155 160 165 Phe Lys Ala Lys
Lys Arg Asp Leu Phe Gly Arg 170 175 <210> SEQ ID NO 4
<211> LENGTH: 568 <212> TYPE: DNA <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 4 ctgcgcactc tatgcatatt
gcagggaaat gattatgttg cgatttttga accaatgttc 60 acaaggccgg
ggcgcgtggc tgttgatggc gtttactgct ctggcactgg aactgacggc 120
gctgtggttc cagcatgtga tgttactgaa accttgcgtg ctctgtattt atgaacgctg
180 cgcgttattc ggcgttctgg gtgctgcgct gattggcgcg atcgccccga
aaactccgct 240 gcgttatgta gcgatggtta tctggttgta tagtgcgttc
cgcggtgtgc agttaactta 300 cgagcacacc atgcttcagc tctatccttc
gccgtttgcc acctgtgatt ttatggttcg 360 tttcccggaa tggctgccgc
tggataagtg ggtgccgcaa gtgtttgtcg cctctggcga 420 ttgcgccgag
cgtcagtggg attttttagg tctggaaatg ccgcagtggc tgctcggtat 480
ttttatcgct tacctgattg tcgcagtgct ggtggtgatt tcccagccgt ttaaagcgaa
540 aaaacgtgat ctgttcggtc gctaataa 568 <210> SEQ ID NO 5
<211> LENGTH: 236 <212> TYPE: PRT <213> ORGANISM:
Escherichia coli <400> SEQUENCE: 5 Met Lys Lys Gly Phe Met
Leu Phe Thr Leu Leu Ala Ala Phe Ser 5 10 15 Gly Phe Ala Gln Ala Asp
Asp Ala Ala Ile Gln Gln Thr Leu Ala 20 25 30 Lys Met Gly Ile Lys
Ser Ser Asp Ile Gln Pro Ala Pro Val Ala 35 40 45 Gly Met Lys Thr
Val Leu Thr Asn Ser Gly Val Leu Tyr Ile Thr 50 55 60 Asp Asp Gly
Lys His Ile Ile Gln Gly Pro Met Tyr Asp Val Ser 65 70 75 Gly Thr
Ala Pro Val Asn Val Thr Asn Lys Met Leu Leu Lys Gln 80 85 90 Leu
Asn Ala Leu Glu Lys Glu Met Ile Val Tyr Lys Ala Pro Gln 95 100 105
Glu Lys His Val Ile Thr Val Phe Thr Asp Ile Thr Cys Gly Tyr 110 115
120 Cys His Lys Leu His Glu Gln Met Ala Asp Tyr Asn Ala Leu Gly 125
130 135 Ile Thr Val Arg Tyr Leu Ala Phe Pro Arg Gln Gly Leu Asp Ser
140 145 150 Asp Ala Glu Lys Glu Met Lys Ala Ile Trp Cys Ala Lys Asp
Lys 155 160 165 Asn Lys Ala Phe Asp Asp Val Met Ala Gly Lys Ser Val
Ala Pro 170 175 180 Ala Ser Cys Asp Val Asp Ile Ala Asp His Tyr Ala
Leu Gly Val 185 190 195 Gln Leu Gly Val Ser Gly Thr Pro Ala Val Val
Leu Ser Asn Gly 200 205 210 Thr Leu Val Pro Gly Tyr Gln Pro Pro Lys
Glu Met Lys Glu Phe 215 220 225 Leu Asp Glu His Gln Lys Met Thr Ser
Gly Lys 230 235 <210> SEQ ID NO 6 <211> LENGTH: 720
<212> TYPE: DNA <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 6 ggaagattta tgaagaaagg ttttatgttg tttactttgt
tagcggcgtt ttcaggcttt 60 gctcaggctg atgacgcggc aattcaacaa
acgttagcca aaatgggcat caaaagcagc 120 gatattcagc ccgcgcctgt
agctggcatg aagacagttc tgactaacag cggcgtgttg 180 tacatcaccg
atgatggtaa acatatcatt caggggccaa tgtatgacgt tagtggcacg 240
gctccggtca atgtcaccaa taagatgctg ttaaagcagt tgaatgcgct tgaaaaagag
300 atgatcgttt ataaagcgcc gcaggaaaaa cacgtcatca ccgtgtttac
tgatattacc 360 tgtggttact gccacaaact gcatgagcaa atggcagact
acaacgcgct ggggatcacc 420 gtgcgttatc ttgctttccc gcgccagggg
ctggacagcg atgcagagaa agaaatgaaa 480 gctatctggt gtgcgaaaga
taaaaacaaa gcgtttgatg atgtgatggc aggtaaaagc 540 gtcgcaccag
ccagttgcga cgtggatatt gccgaccatt acgcacttgg cgtccagctt 600
ggcgttagcg gtactccggc agttgtgctg agcaatggca cacttgttcc gggttaccag
660 ccgaaagaga tgaaagaatt cctcgacgaa caccaaaaaa tgaccagcgg
taaataataa 720 <210> SEQ ID NO 7 <211> LENGTH: 489
<212> TYPE: PRT <213> ORGANISM: Escherichia coli
<400> SEQUENCE: 7 Met Gln Leu Pro Gln Gly Val Trp His Glu Asp
Glu Phe Tyr Gly 5 10 15 Lys Ser Glu Ile Tyr Arg Asp Arg Leu Thr Leu
Pro Val Thr Ile 20 25 30 Asn Gln Ala Ser Ala Gly Ala Thr Leu Thr
Val Thr Tyr Gln Gly 35 40 45 Cys Ala Asp Ala Gly Phe Cys Tyr Pro
Pro Glu Thr Lys Thr Val 50 55 60 Pro Leu Ser Glu Val Val Ala Asn
Asn Ala Ala Pro Gln Pro Val 65 70 75 Ser Val Pro Gln Gln Glu Gln
Pro Thr Ala Gln Leu Pro Phe Ser 80 85 90 Ala Leu Trp Ala Leu Leu
Ile Gly Ile Gly Ile Ala Phe Thr Pro 95 100 105 Cys Val Leu Pro Met
Tyr Pro Leu Ile Ser Gly Ile Val Leu Gly 110 115 120 Gly Lys Gln Arg
Leu Ser Thr Ala Arg Ala Leu Leu Leu Thr Phe 125 130 135 Ile Tyr Val
Gln Gly Met Ala Leu Thr Tyr Thr Ala Leu Gly Leu 140 145 150 Val Val
Ala Ala Ala Gly Leu Gln Phe Gln Ala Ala Leu Gln His 155 160 165 Pro
Tyr Val Leu Ile Gly Leu Ala Ile Val Phe Thr Leu Leu Ala 170 175 180
Met Ser Met Phe Gly Leu Phe Thr Leu Gln Leu Pro Ser Ser Leu 185 190
195 Gln Thr Arg Leu Thr Leu Met Ser Asn Arg Gln Gln Gly Gly Ser 200
205 210 Pro Gly Gly Val Phe Val Met Gly Ala Ile Ala Gly Leu Ile Cys
215 220 225 Ser Pro Cys Thr Thr Ala Pro Leu Ser Ala Ile Leu Leu Tyr
Ile 230 235 240 Ala Gln Ser Gly Asn Met Trp Leu Gly Gly Gly Thr Leu
Tyr Leu 245 250 255 Tyr Ala Leu Gly Met Gly Leu Pro Leu Met Leu Ile
Thr Val Phe 260 265 270 Gly Asn Arg Leu Leu Pro Lys Ser Gly Pro Trp
Met Glu Gln Val 275 280 285 Lys Thr Ala Phe Gly Phe Val Ile Leu Ala
Leu Pro Val Phe Leu 290 295 300 Leu Glu Arg Val Ile Gly Asp Val Trp
Gly Leu Arg Leu Trp Ser 305 310 315 Ala Leu Gly Val Ala Phe Phe Gly
Trp Ala Phe Ile Thr Ser Leu 320 325 330 Gln Ala Lys Arg Gly Trp Met
Arg Ile Val Gln Ile Ile Leu Leu 335 340 345 Ala Ala Ala Leu Val Ser
Val Arg Pro Leu Gln Asp Trp Ala Phe 350 355 360 Gly Ala Thr His Thr
Ala Gln Thr Gln Thr His Leu Asn Phe Thr 365 370 375 Gln Ile Lys Thr
Val Asp Glu Leu Asn Gln Ala Leu Val Glu Ala 380 385 390 Lys Gly Lys
Pro Val Met Leu Asp Leu Tyr Ala Asp Trp Cys Val 395 400 405 Ala Cys
Lys Glu Phe Glu Lys Tyr Thr Phe Ser Asp Pro Gln Val 410 415 420 Gln
Lys Ala Leu Ala Asp Thr Val Leu Leu Gln Ala Asn Val Thr 425 430 435
Ala Asn Asp Ala Gln Asp Val Ala Leu Leu Lys His Leu Asn Val 440 445
450 Leu Gly Leu Pro Thr Ile Leu Phe Phe Asp Gly Gln Gly Gln Glu 455
460 465 His Pro Gln Ala Arg Val Thr Gly Phe Met Asp Ala Glu Thr Phe
470 475 480 Ser Ala His Leu Arg Asp Arg Gln Pro 485 <210> SEQ
ID NO 8 <211> LENGTH: 1474 <212> TYPE: DNA <213>
ORGANISM: Escherichia coli <400> SEQUENCE: 8 cgtgcagctg
ccgcaaggcg tctggcatga agatgagttt tacggcaaaa gcgagattta 60
ccgcgatcgg ctgacgcttc ccgtcaccat caaccaggcg agtgcgggag cgacgttaac
120 tgtcacctac cagggctgtg ctgatgccgg tttctgttat ccgccagaaa
ccaaaaccgt 180 tccgttaagc gaagtggtcg ccaacaacgc agcgccacag
cctgtgtctg ttccgcagca 240 agagcagccc accgcgcaat tgcccttttc
cgcgctctgg gcgttgttga tcggtattgg 300 tatcgccttt acgccatgcg
tgctgccaat gtacccactg atttctggca tcgtgctggg 360 tggtaaacag
cggctctcca ctgccagagc attgttgctg acctttattt atgtgcaggg 420
gatggcgctg acctacacgg cgctgggtct ggtggttgcc gccgcagggt tacagttcca
480 ggcggcgcta cagcacccat acgtgctcat tggcctcgcc atcgtcttta
ccttgctggc 540 gatgtcaatg tttggcttgt ttaccctgca actcccctct
tcgctgcaaa cacgtctcac 600 gttgatgagc aatcgccaac agggcggctc
acctggcggt gtgtttgtta tgggggcgat 660 tgccggactg atctgttcac
catgcaccac cgcaccgctt agcgcgattc tgctgtatat 720 cgcccaaagc
gggaacatgt ggctgggcgg cggcacgctt tatctctatg cgttgggcat 780
gggcctgccg ctgatgctaa ttaccgtctt tggtaaccgc ttgctgccga aaagcggccc
840 gtggatggaa caagtcaaaa ccgcgtttgg ttttgtgatc ctcgcactgc
cggtcttcct 900 gctggagcga gtgattggtg atgtatgggg attacgcttg
tggtcggcgc tgggtgtcgc 960 attctttggc tgggccttta tcaccagcct
acaggctaaa cgcggctgga tgcgtattgt 1020 gcaaattatt ctgctggcag
cggcattggt tagcgtgcgc ccacttcagg attgggcatt 1080 tggtgcgacg
cataccgcgc aaactcagac gcatctcaac tttacacaaa tcaaaacggt 1140
agatgagtta aatcaggcgc tcgttgaagc caaaggcaaa ccggtgatgt tagatcttta
1200 tgccgactgg tgcgtcgcct gtaaagagtt tgagaaatac accttcagcg
acccgcaggt 1260 gcaaaaagcg ttagcagaca cggtcttact tcaggccaac
gtcacggcca acgacgcaca 1320 agatgtggcg ctgttaaagc atcttaatgt
ccttggccta ccgacaattc tcttttttga 1380 cggacaaggc caggagcatc
cacaagcacg cgtcacgggc tttatggatg ctgaaacctt 1440 cagcgcacat
ttgcgcgatc gccaaccgtg ataa 1474
<210> SEQ ID NO 9 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <400> SEQUENCE:
9 cggagctcat cggagagagt aga 23 <210> SEQ ID NO 10 <211>
LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <400> SEQUENCE: 10 ggcccgggaa ttattatttt ttctcgga 28
<210> SEQ ID NO 11 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <400> SEQUENCE:
11 ggcccgggct gcgcactcta tgcatattgc aggg 34 <210> SEQ ID NO
12 <211> LENGTH: 34 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <400> SEQUENCE: 12 ggcatatgga
ttattagcga ccgaacagat cacg 34 <210> SEQ ID NO 13 <211>
LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <400> SEQUENCE: 13 ggcatatgag gaggaagatt tatgaagaaa
gg 32 <210> SEQ ID NO 14 <211> LENGTH: 44 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <400>
SEQUENCE: 14 ccgtcgacga ttattattta ccgctggtca ttttttggtg ttcg 44
<210> SEQ ID NO 15 <211> LENGTH: 42 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <400> SEQUENCE:
15 ccgtcgacga ggccgacatg cagctgccgc aaggcgtctg gc 42 <210>
SEQ ID NO 16 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <400> SEQUENCE: 16
ccgcatgctt atcacggttg gcgatcgcgc 30 <210> SEQ ID NO 17
<211> LENGTH: 3457 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <400> SEQUENCE: 17 gagctcatcg
gagagagtag atcatgaaaa agatttggct ggcgctggct ggtttagttt 60
tagcgtttag cgcatcggcg gcgcagtatg aagatggtaa acagtacact accctggaaa
120 aaccagttgc tggcgcgccg caagtgctgg agtttttctc tttcttctgc
ccgcactgct 180 atcagtttga agaagttctg catatttctg ataacgtgaa
gaaaaaactg ccggaaggcg 240 tgaagatgac taaataccac gtcaacttca
tggggggtga cctgggcaaa gagctgactc 300 aggcatgggc tgtggcgatg
gcgctgggcg tggaagacaa agtcacagtt ccgctgtttg 360 aaggcgtaca
aaaaacccag accattcgtt cagcatctga tatccgcgat gtatttatca 420
acgcaggtat taaaggtgaa gagtacgacg cggcgtggaa cagcttcgtg gtgaaatctc
480 tggtcgctca gcaggaaaaa gctgcagctg acgtgcaatt gcgtggtgtt
ccggcgatgt 540 ttgttaacgg taaatatcag ctgaatccgc agggtatgga
taccagcaat atggatgttt 600 ttgttcagca gtatgctgat actgtgaaat
atctgtccga gaaaaaataa taattcccgg 660 gctgcgcact ctatgcatat
tgcagggaaa tgattatgtt gcgatttttg aaccaatgtt 720 cacaaggccg
gggcgcgtgg ctgttgatgg cgtttactgc tctggcactg gaactgacgg 780
cgctgtggtt ccagcatgtg atgttactga aaccttgcgt gctctgtatt tatgaacgct
840 gcgcgttatt cggcgttctg ggtgctgcgc tgattggcgc gatcgccccg
aaaactccgc 900 tgcgttatgt agcgatggtt atctggttgt atagtgcgtt
ccgcggtgtg cagttaactt 960 acgagcacac catgcttcag ctctatcctt
cgccgtttgc cacctgtgat tttatggttc 1020 gtttcccgga atggctgccg
ctggataagt gggtgccgca agtgtttgtc gcctctggcg 1080 attgcgccga
gcgtcagtgg gattttttag gtctggaaat gccgcagtgg ctgctcggta 1140
tttttatcgc ttacctgatt gtcgcagtgc tggtggtgat ttcccagccg tttaaagcga
1200 aaaaacgtga tctgttcggt cgctaataat ccatatgagg aggaagattt
atgaagaaag 1260 gttttatgtt gtttactttg ttagcggcgt tttcaggctt
tgctcaggct gatgacgcgg 1320 caattcaaca aacgttagcc aaaatgggca
tcaaaagcag cgatattcag cccgcgcctg 1380 tagctggcat gaagacagtt
ctgactaaca gcggcgtgtt gtacatcacc gatgatggta 1440 aacatatcat
tcaggggcca atgtatgacg ttagtggcac ggctccggtc aatgtcacca 1500
ataagatgct gttaaagcag ttgaatgcgc ttgaaaaaga gatgatcgtt tataaagcgc
1560 cgcaggaaaa acacgtcatc accgtgttta ctgatattac ctgtggttac
tgccacaaac 1620 tgcatgagca aatggcagac tacaacgcgc tggggatcac
cgtgcgttat cttgctttcc 1680 cgcgccaggg gctggacagc gatgcagaga
aagaaatgaa agctatctgg tgtgcgaaag 1740 ataaaaacaa agcgtttgat
gatgtgatgg caggtaaaag cgtcgcacca gccagttgcg 1800 acgtggatat
tgccgaccat tacgcacttg gcgtccagct tggcgttagc ggtactccgg 1860
cagttgtgct gagcaatggc acacttgttc cgggttacca gccgaaagag atgaaagaat
1920 tcctcgacga acaccaaaaa atgaccagcg gtaaataata atcgtcgacg
aggccgacat 1980 gcagctgccg caaggcgtct ggcatgaaga tgagttttac
ggcaaaagcg agatttaccg 2040 cgatcggctg acgcttcccg tcaccatcaa
ccaggcgagt gcgggagcga cgttaactgt 210 cacctaccag ggctgtgctg
atgccggttt ctgttatccg ccagaaacca aaaccgttcc 2160 gttaagcgaa
gtggtcgcca acaacgcagc gccacagcct gtgtctgttc cgcagcaaga 2220
gcagcccacc gcgcaattgc ccttttccgc gctctgggcg ttgttgatcg gtattggtat
2280 cgcctttacg ccatgcgtgc tgccaatgta cccactgatt tctggcatcg
tgctgggtgg 2340 taaacagcgg ctctccactg ccagagcatt gttgctgacc
tttatttatg tgcaggggat 2400 ggcgctgacc tacacggcgc tgggtctggt
ggttgccgcc gcagggttac agttccaggc 2460 ggcgctacag cacccatacg
tgctcattgg cctcgccatc gtctttacct tgctggcgat 2520 gtcaatgttt
ggcttgttta ccctgcaact cccctcttcg ctgcaaacac gtctcacgtt 2580
gatgagcaat cgccaacagg gcggctcacc tggcggtgtg tttgttatgg gggcgattgc
2640 cggactgatc tgttcaccat gcaccaccgc accgcttagc gcgattctgc
tgtatatcgc 2700 ccaaagcggg aacatgtggc tgggcggcgg cacgctttat
ctctatgcgt tgggcatggg 2760 cctgccgctg atgctaatta ccgtctttgg
taaccgcttg ctgccgaaaa gcggcccgtg 2820 gatggaacaa gtcaaaaccg
cgtttggttt tgtgatcctc gcactgccgg tcttcctgct 2880 ggagcgagtg
attggtgatg tatggggatt acgcttgtgg tcggcgctgg gtgtcgcatt 2940
ctttggctgg gcctttatca ccagcctaca ggctaaacgc ggctggatgc gtattgtgca
3000 aattattctg ctggcagcgg cattggttag cgtgcgccca cttcaggatt
gggcatttgg 3060 tgcgacgcat accgcgcaaa ctcagacgca tctcaacttt
acacaaatca aaacggtaga 3120 tgagttaaat caggcgctcg ttgaagccaa
aggcaaaccg gtgatgttag atctttatgc 3180 cgactggtgc gtcgcctgta
aagagtttga gaaatacacc ttcagcgacc cgcaggtgca 3240 aaaagcgtta
gcagacacgg tcttacttca ggccaacgtc acggccaacg acgcacaaga 3300
tgtggcgctg ttaaagcatc ttaatgtcct tggcctaccg acaattctct tttttgacgg
3360 acaaggccag gagcatccac aagcacgcgt cacgggcttt atggatgctg
aaaccttcag 3420 cgcacatttg cgcgatcgcc aaccgtgata agcatgc 3457
<210> SEQ ID NO 18 <211> LENGTH: 64 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Duplex is formed in the region from
position 5 to 60 of the base sequence. <400> SEQUENCE: 18
agctcgcgaa gcttgcatgc tgcagtcgac atatgcccgg gtaccgagct cgcggccgca
60 tgca 64 <210> SEQ ID NO 19 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<400> SEQUENCE: 19 Met Lys Lys Thr Ala Ile Ala Ile Ala Val
Ala Leu Ala Gly Phe 1 5 10 15 Ala Thr Val Ala Asn Ala 20
<210> SEQ ID NO 20 <211> LENGTH: 73 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <400> SEQUENCE:
20 atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac
cgtagcgcag 60 gccggctgaa ttc 73 <210> SEQ ID NO 21
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <400> SEQUENCE: 21 Met Arg Ala Lys Leu
Leu Gly Ile Val Leu Thr Thr Pro Ile Ala 1 5 10 15 Ile Ser Ser Phe
Ala 20 <210> SEQ ID NO 22 <211> LENGTH: 70 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <400>
SEQUENCE: 22 atgcgcgcga aactgctggg tattgtcctg acgaccccga tcgcgatcag
ctcttttgcc 60 ggctgaattc 70 <210> SEQ ID NO 23 <211>
LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <400> SEQUENCE: 23 agcagctccc atccgatctt ccaccgcggc
gaattc 36 <210> SEQ ID NO 24 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<400> SEQUENCE: 24 atgcagttaa cccctacatt c 21 <210> SEQ
ID NO 25 <211> LENGTH: 21 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <400> SEQUENCE: 25 ggggaattcg
gatccttatt a 21
* * * * *